2232
Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates
the cytotoxic activity of temozolomide in leukemia cells:
influence of mismatch repair status and O6-methylguanineDNA methyltransferase activity
Terzah M. Horton,1 Gaye Jenkins,1
Debananda Pati,1 Linna Zhang,1 M. Eileen Dolan,2
Albert Ribes-Zamora,1 Alison A. Bertuch,1
Susan M. Blaney,1 Shannon L. Delaney,2
Madhuri Hegde,3 and Stacey L. Berg1
Texas Children's Cancer Center/Baylor College of Medicine,
Houston, Texas; 2Department of Medicine and Cancer Research
Center, University of Chicago, Chicago, Illinois; and 3Department
of Human Genetics, Emory University School of Medicine,
Atlanta, Georgia
hanced TMZ activity in MMR-proficient cells (PF = 3–7).
ABT-888 potentiation was unrelated to NHEJ activity.
ABT-888 potentiated TMZ (PF = 2–5) in two of four acute
myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most
pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression
completely abrogated ABT-888 potentiation of TMZ. [Mol
Cancer Ther 2009;8(8):2232–42]
Abstract
Introduction
The poly(ADP-ribose) polymerase (PARP) inhibitor ABT888 potentiates the antitumor activity of temozolomide
(TMZ). TMZ resistance results from increased O6-methylguanine-DNA methyltransferase (MGMT) activity and
from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP
activity in ABT-888 potentiation of TMZ. MMR-proficient
and MMR-deficient leukemia cells with varying MGMT
activity, as well as primary leukemia samples, were used
to determine TMZ IC50 alone and with ABT-888. ABT-888
effectively inhibited PARP activity and enhanced TMZ
growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low
MGMT activity [potentiation factor (PF) = 21]. ABT-888
also potentiated TMZ activity in MMR-deficient cells with
elevated MGMT activity. Unexpectedly, ABT-888 also en-
DNA repair systems mediate tumor cell response to DNAdamaging anticancer agents. Temozolomide (TMZ), an alkylating agent active in the treatment of gliomas (1, 2) and
some leukemias (3, 4), creates DNA damage by adding
methyl adducts to N7 guanine (70% total adducts), N3 adenine (9%), and O6 guanine (5%; Fig. 1; ref. 5). The cytotoxicity of TMZ has been attributed to the creation of
O 6-methylguanine (6), which results in single-stranded
DNA breaks, growth arrest, and apoptosis (7, 8). TMZ resistance results from at least two mechanisms: (a) increased
levels of the DNA repair protein O6-methylguanine-DNA
methyltransferase (MGMT), which removes O6-methyl adducts from the O6 position of guanine in DNA, and (b) deficiencies in the DNA mismatch repair (MMR) system,
resulting in microsatellite instability (MSI) and tolerance of
O6-methylgaunine adduct DNA mismatches (Fig. 1; ref. 9).
Although MGMT inhibitors, such as O6-benzylguanine
(O6-BG), can effectively overcome TMZ resistant in MMRproficient (MSI stable) cells, they are ineffective in cells with
MMR deficiencies (8, 10).
Because TMZ also generates N-methylated bases (N3 and
7
N ), which can be removed by the base excision repair (BER)
system (5), robust BER activity can result in TMZ resistance
(5, 11). Central to BER and the removal of methylated N3 and
N 7 adducts is the enzyme poly(ADP-ribose) polymerase
(PARP), an abundant nuclear enzyme that senses both
single-stranded DNA and dsDNA breaks. In BER, PARP acts
as a nick sensor, catalyzing the cleavage of NAD+ and attaching PARP to itself, histones, and other target proteins. Negatively charged ADP-ribose polymers create electrostatic
repulsions between DNA and histones, opening chromatin
for DNA repair; PARP also recruits BER proteins to sites of
single-stranded DNA breaks, initiating DNA repair (12).
Thus, PARP inhibitors (PARPi) may overcome TMZ resistance in MMR-deficient cells by blocking BER, resulting in
cytotoxicity from N3- and N7-methyl adducts (11, 13).
1
Received 2/16/09; revised 5/6/09; accepted 6/2/09; published
OnlineFirst 8/11/09.
Grant support: K12 CA90433-04 (T.M. Horton), K23 CA113775 (T.M.
Horton), Scott Carter NCCF Research Fellowship (T.M. Horton), Ladies
Leukemia League (T.M. Horton), RO1 CA109478 (D. Pati), RO1 CA81485
(M.E. Dolan), and U01 CA63187 (University of Chicago Cancer Research
Center). Pharmacologic studies were supported by the University of
Chicago Cancer Research Center Pharmacology Core Facility (http://
pharmacology.bsd.uchicago.edu/) through the University of Chicago
Cancer Research Center Cancer Center Support Grant P30 CA14599.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
Note: Supplementary material for this article is available at Molecular
Cancer Therapeutics Online (http://mct.aacrjournals.org/).
Requests for reprints: Terzah M. Horton, Baylor College of Medicine, 6621
Fannin, MC 3-3320, Houston, TX 77030. Phone: 832-824-4269;
Fax: 832-825-4276. E-mail: tmhorton@txccc.org
Copyright © 2009 American Association for Cancer Research.
doi:10.1158/1535-7163.MCT-09-0142
Mol Cancer Ther 2009;8(8). August 2009
Molecular Cancer Therapeutics
PARP inhibitors have been tested in several tumor types
and have been shown to enhance the antitumor effects of
TMZ in leukemia (13), glioma (14–16), lung (17, 18), and
colon carcinoma, both in vitro (16, 18–20) and in xenograft
models (17, 21). Previous research has shown that the oral
PARPi ABT-888 effectively inhibits PARP activity in animals
(22, 23). In a phase 0 trial in humans, a single 25 mg dose
of ABT-888 resulted in a median plasma ABT-888 concentration of 210 nmol/L, resulting in >92% PARP inhibition (24).
Because MMR status has been well characterized in a
wide range of established leukemia cell lines, our objective
was to use these cell lines as a model to assess the relative
importance of MGMT activity and MMR status on the ability of ABT-888 to potentiate the growth-inhibitory effects of
TMZ. ABT-888 has previously been shown to inhibit both
PARP-1 and PARP-2 isoenzymes (22). Our goal was to determine (a) whether PARPi potentiation of TMZ was effective in cells with MMR proficiency, (b) whether PARPi
potentiation of TMZ was abrogated by elevated MGMT,
and (c) whether other mechanisms influence PARPi potentiation of TMZ.
Materials and Methods
Chemicals
RPMI 1640 cell culture medium, PBS, dextrose, sodium
pyruvate, sodium bicarbonate, and HEPES were purchased
from Life Technologies; fetal calf serum and high-glucose
RPMI 1640 cell culture medium were purchased from the
American Type Culture Collection; bovine growth serum
was purchased from Hyclone; penicillin/streptomycin was
purchased from Invitrogen; and Lymphoprep for mononuclear cell isolation was purchased from Greiner Bio-One.
ABT-888 was synthesized and kindly provided by Abbott
Laboratories. ABT-888 was diluted in DMSO to a stock concentration of 62 mmol/L. O6-BG (NSC 637037) was provided
by the Cancer Therapy and Evaluation Program of the
National Cancer Institute. TMZ (Schering-Plough) was purchased and formulated in DMSO according to the manufacturers' recommendations.
Cell Lines
The human T-cell acute lymphoblastic leukemia (ALL)
cell lines Jurkat, Molt4, and HSB2; the human pre-B ALL
cell lines JM1 and Reh; the B-cell lines Raji and Daudi; the
histiocytic cell line U937; and the acute myeloid leukemia
(AML) cell lines HL-60 (acute promyelocytic leukemia),
KG1, HEL (erythroleukemia), and THP1 (monocytic leukemia) were purchased and cultured as directed by the American Type Culture Collection.
Culture of Primary Leukemia Cells
Leukemia cells were obtained from peripheral blood, leukapheresis, or bone marrow aspirate specimens from children with newly diagnosed acute leukemia before
chemotherapy in accordance with Institutional Review
Board guidelines. Peripheral blood mononuclear cells were
isolated using Lymphoprep and frozen at a cell density of
1 × 107/mL at −80°C until use. Primary leukemia cells were
Mol Cancer Ther 2009;8(8). August 2009
cultured in RPMI 1640 supplemented with 20% FCS and
penicillin/streptomycin. During drug sensitivity assays, cell
viability was determined by trypan blue exclusion at 48 or
72 h and noted to be >90% in the absence of drug.
In vitro Cytotoxicity Assays
The growth inhibition effect of ABT-888 and TMZ was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric dye reduction as
previously described (25) or the CellTiter-Glo luminescent
cell viability assay (Promega) according to the manufacturer's instructions. Leukemia cell lines were plated at a cell
density of 0.5 to 2 × 105/mL. TMZ was serially diluted over
a 106-fold range of concentrations to determine single-agent
IC50s as described (4). For assays assessing single drug activity or TMZ in combination with ABT-888, replicates of
six-wells were used for each drug concentration and the
assay was repeated using two replicate plates. ABT-888
was tested in MMR-proficient U937, THP1, and JM1 and
in MMR-deficient HSB2, Molt4, Jurkat, and Reh (Table 1).
IC50 values for each cell line were determined in at least
three independent experiments using the Hill equation as
previously described (25). Primary cells were plated at a
concentration of 8 to 40 × 104/mL and drug concentrations
were tested in triplicate. IC50 values for each primary cell
sample were determined in at least two independent experiments. In combination experiments, ABT-888 and/or O6-BG
were added 30 min before TMZ. Viability was assessed after
72 h. Representative cell lines combining TMZ with ABT-888
are shown from each MGMT and MSI subgroup (Fig. 3A
and B). Combinations between TMZ, O6-BG, and ABT-888
were tested in the MMR-proficient cell lines JM1 and U937
as well as the MMR-deficient cell lines Jurkat and Molt4.
The combination effect of drugs used with TMZ (O6-BG
and ABT-888) was modeled using the universal response
surface approach method as described (25, 26).
MGMT Activity, MSI, and PARP Activity Assays
MGMT activity in peripheral blood mononuclear cell or
bone marrow aspirate tumor cell lysates was determined
by the removal of O6-[3H]methylguanine from a 3H-methylated DNA substrate and quantified as fmol O6-[3H]methylguanine/mg total protein as described (27). For MSI
determination in four representative primary leukemia cells
(patients p115 and p120 with AML and patients p152 and
p157 with ALL), genomic DNA was extracted and amplified
using three MSI multiplex reaction mixtures containing National Cancer Institute panel markers (BAT-25, BAT-26,
D2S123, D17S250, and D5S346; ref. 28), quasimonomorphic
mononucleotide markers (NR-21, NR-22, NR-24, BAT-25,
and BAT-26; ref. 29), or an alternative panel of markers
(D18S35, TP53-DI, TP53-PENTA, D1S2883, and FGA) as described (4). MSI status of established cell lines was obtained
from the literature (9, 30–33). PARP activity was measured
by the incorporation of biotinylated poly(ADP-ribose) into
histones using modifications of a commercially available
PARP assay (Trevigen). Cell lysates were prepared from
1 × 107 leukemia cells using 1× PARP buffer (Trevigen) supplemented with 0.4 mmol/L phenylmethylsulfonyl fluoride, one-half complete protease inhibitor cocktail tablet
2233
2234 ABT-888 and Temozolomide in Acute Leukemia
Figure 1. Model of the effects of PARP inhibition
on TMZ activity. TMZ creates O6-methylguanine adducts (CH2) and N7- and N3-methyl adducts (CH3 on
either adenine or guanine), which are efficiently removed by BER. Model shows guanine as a representative nucleotide. TMZ resistance results from either
(a) MMR mutations (left), which allow DNA replication in the presence of dinucleotide (mG-T) mismatches, or (b) elevated MGMT activity (right),
which removes the O6-methyl group from guanine.
PARP inhibition blocks the repair of N3- and N7-methyl
adducts, resulting in apoptosis during cell division.
(Roche), 1% NP40, and 0.1% SDS. Each sample was done in
triplicate, and recombinant PARP (Trevigen) was used to
produce a standard curve for each assay. PARP activity
was assessed in either duplicate or triplicate in at least
two independent experiments. PARP inhibition with ABT888 was determined in MMR-proficient cell lines JM1 and
U937 as well as MMR-deficient cell lines Molt4, Reh, Jurkat,
and HSB2.
Immunoblots
Cell lysates were prepared from 1 × 107 cells as previously
described (34). Thirty to 50 μg of protein extract from representative cell lines (MMR-proficient cell lines JM1, U937,
and THP1 as well as MMR-deficient cell lines Molt4, Reh,
HSB2, and Jurkat) were run on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). Membranes
were incubated overnight with MGMT (1:500 dilution; Cell
Signaling Technology), 1 μg/mL anti-human PARP (BD
Pharmingen), 4 μg/mL anti-Ku70 (clone N3H10; Abcam),
rabbit anti-human phospho-histone H2AX (1:1,000 dilution;
Cell Signaling Technology), or β-actin (1:10,000 dilution;
Sigma) antibodies diluted in Odyssey blocking buffer
(Li-Cor). Bound primary antibodies were detected with
IR-800 or IR-700 dye-labeled, appropriate species-specific
secondary antisera and visualized on a Li-Cor Odyssey IR
scanner. The intensity of gel bands was measured using
Molecular Dynamics ImageQuant software (version 5.2).
Nonhomologous End-Joining Assay
Small-scale in vitro nonhomologous end-joining (NHEJ)
assays were done essentially as described (35), except that
a 2.9-kb PvuII- and HindIII-linearized 5′- 32 P-labeled
pCDNA3.1 fragment was used. Reactions (10 μL) were done
with 30 μg of protein extract and 10 ng of 32P-labeled DNA.
Full image is provided in Supplementary Fig. S1.
Transient Transfection Assay
pCDNA3.3 and pcDNA-MGMT plasmids were transfected into MMR-proficient THP1 AML cells and MMRdeficient HSB2 T-cell ALL cells using Nucleofector Kit V
from Amaxa, Inc. as directed with minor modifications.
Approximately 1.5 × 106 cells were suspended in 100 μL
of Nucleofector Solution V containing 0.5 μg of plasmid
DNA. Plasmid pEGFP was used to monitor transfection
efficiency. Following 6 to 8 h of incubation at 37°C, cells
Mol Cancer Ther 2009;8(8). August 2009
Molecular Cancer Therapeutics
were washed and cultured for another 40 h before protein
lysates were made and cytotoxicity assays were done. The
transfection efficiency, measured by counting the green fluorescent cells, was estimated to be 70% to 80% for each of the
cell lines used (full image in Supplementary Fig. S1)
Statistics
Dose-response curves are shown using mean ± SD. IC50s
were calculated using the Hill equation as previously described (25). MGMT activity and PARP activity are reported
as mean ± SD of each sample done in triplicate. PARP activity in MMR-proficient and MMR-deficient cell lines was
compared using a two-sided Student's t test. PARP activity,
MGMT activity, and potentiation factors (PF) were compared as continuous variables using the Wilcoxon ranksum test. K50s and PF were determined from at least three
independent experiments.
Results
TMZ and PARP Inhibition in Leukemia Cell Lines
The growth-inhibitory effects of TMZ and the PARPi
ABT-888 were tested in MMR-proficient and MMR-deficient
leukemia cell lines with variable MGMT activity (Table 1).
Previous work has shown that plasma TMZ concentrations
(Cmax) reached ∼62 μmol/L when given at the maximum
tolerated dose (200 mg/m2) over 5 days (36). As shown in
Fig. 2A, most leukemia cell lines were resistant to TMZ at
this concentration. Although three MMR-proficient cell lines
(JM1, HEL, and U937) were more sensitive to TMZ (IC50,
11–50 μmol/L; Fig. 2A), MMR-deficient cell lines were resistant to TMZ, with an average IC50 of 450 μmol/L. Leukemia
cells with elevated MGMT activity (Jurkat, Reh, and KG1)
were also more resistant to TMZ than cell lines with absent
(U937 and HEL) or low (JM1) MGMT activity.
ABT-888 also inhibited leukemia cell growth, with IC50s
ranging from 20 to 196 μmol/L (Fig. 2B). These ABT-888 inhibitory concentrations, however, were approximately 5- to
33-fold higher than plasma concentrations achieved in either animals or humans (22, 24). Because steady-state concentrations in animals ranged from 0.35 to 1 μmol/L, and
the single-dose ABT-888 Cmax was 0.2 to 11 μmol/L, further
exploration of the ABT-888–potentiating effects was done
using concentrations of 0.5 and 5 μmol/L.
V a r i a b l e M G M T A c t i vi t y a n d M M R S t a t u s i n
Leukemia Cell Lines
MGMT expression is variable in primary leukemia cells,
ranging from undetectable to 6,000 fmol/mg protein (4).
As shown in Table 1, MGMT activity varied in both
MMR-proficient (MSI stable) and MMR-deficient (MSI unstable) leukemia cell lines. In MMR-proficient cells, MGMT
activity varied from undetectable in the AML cell lines HEL
and U937 to high (1,170 fmol/mg protein) in the AML cell
line KG1. In MMR-deficient (MSI unstable) cell lines,
MGMT activity ranged from 320 fmol/mg protein in the
T-cell ALL line HSB2 to 1,300 fmol/mg protein in the preB ALL cell line Reh. To assess the relative contributions of
MGMT activity and MMR status to ABT-888 potentiation of
TMZ, leukemia cell lines were subdivided into seven groups
based on relative MGMT activity and MMR status (Table 1).
Because normal adult peripheral blood mononuclear cells
express an average of 770 + 170 fmol/mg protein MGMT
activity (4), MMR-proficient cell lines (MSI stable; S) were
divided into those with undetectable (S0), low (S1), average
(S2), or high (S3) MGMT activity, corresponding to undetectable, <460 fmol/mg, 460 to 1,100 fmol/mg, and
>1,110 fmol/mg MGMT protein, respectively. MMRdeficient cell lines (MSI unstable; U) were similarly classified into groups with low (U1), average (U2), and high
(U3) MGMT activity based on the same criteria.
ABT-888 Potentiates TMZ Activity in Cells with
Proficient MMR and MGMT Activity
With one exception (U937 AML cells), ABT-888 enhanced
the activity of TMZ in MMR-proficient cells from 3- to
7-fold, an effect that seemed to be independent of MGMT
activity (Fig. 3A and C). ABT-888 was more effective in potentiating TMZ-induced cytotoxicity in MMR-deficient cells
(10- to 21-fold; Fig. 3B) and was most potent in MMRdeficient leukemia cells with low MGMT activity (U1), such
as the T-cell ALL line HSB2 (Fig. 3B, left). In this cell line,
ABT-888 decreased the TMZ IC50 from 440 to 20 μmol/L,
Table 1. Summary of leukemia subtype, MGMT activity, and MSI status in leukemia cell lines
Cell line
U937
HEL
THP1
JM1
Raji
HL-60
Daudi
KG1
HSB2
Molt4
Jurkat
Reh
Leukemia subtype
MGMT activity (fmol/mg protein)
MSI
Group*
AML
AML
AML
Pre-B ALL
B ALL
AML
B ALL
AML
T-ALL
T-ALL
T-ALL
Pre-B ALL
Absent
Absent
Low (370 ± 11)
Low (450 ± 17)
Average (380 ± 89)
Average (750 ± 94)
Average (902 ± 17)
High (1,170 ± 34)
Low (320 ± 20)
Average (560 ± 37)
High (1,290 ± 36)
High (1,300 ± 14)
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Stable
Unstable
Unstable
Unstable
Unstable
S0
S0
S1
S1
S1
S2
S2
S3
U1
U2
U3
U3
*Group assignment based on MGMT activity (0 = none, 1 = low, 2 = medium, 3 = high) and MSI status (S = stable, U = unstable).
Mol Cancer Ther 2009;8(8). August 2009
2235
2236 ABT-888 and Temozolomide in Acute Leukemia
a 21-fold enhancement. ABT-888 alone had no effect
on HSB2 growth at the same concentration (5 μmol/L;
Fig. 2B). ABT-888 also enhanced the effects of TMZ in
MMR-deficient cell lines with average or elevated MGMT activity. In pre-B and T-ALL cell lines with the highest MGMT
activity (U3), ABT-888 provided a 10- to 13-fold potentiation
(Fig. 3B, right). This suggests that ABT-888 can enhance TMZinduced cytotoxicity in MMR-deficient leukemia cell lines
despite elevated MGMT activity.
Combination of MGMT Inhibitor O6-BG with ABT-888
and TMZ
Because MGMT inhibitors have been extensively studied
as a means to increase TMZ efficacy (37, 38), it was of interest to determine the relative potentiation of the MGMT inhibitor O6-BG and ABT-888. In MMR-proficient cell lines,
such as JM1, O6-BG seemed to provide more TMZ potentiation than ABT-888 (Fig. 3D) and ABT-888 did not provide
any additional TMZ potentiation. In MMR-deficient cell
lines, however, such as the T-cell ALL cell lines Molt4
(Fig. 3E) and Jurkat (data not shown), the addition of both
O6-BG and ABT-888 each potentiated TMZ and the addition
of both O6-BG and ABT-888 to TMZ was additive when
modeled using the universal response surface approach
method (26).
Overexpression of MGMT Does Not Prevent ABT-888
Potentiation of TMZ
To determine if MGMT overexpression could overcome
ABT-888 potentiation of TMZ growth inhibition, we transfected the MMR-proficient THP1 and MMR-deficient HSB2
cells with a construct constitutively expressing MGMT. By
immunoblot densitometry, MGMT expression was increased at least 30-fold (Fig. 4A). Increase in MGMT expression was able to partially, but not completely, overcome
ABT-888 potentiation of TMZ cytotoxicity. In the MMRproficient THP1 cells (Fig. 4B), PF decreased from 3.4
to 1.6, and in the MMR-deficient cell lines HSB2 (Fig. 4C),
the PF decreased from 15 to 5.8.
ABT-888 Potentiation of TMZ: Relationship to PARP
Activity
To determine whether ABT-888–mediated TMZ potentiation was related to the degree of PARP inhibition, immunoblots and activity assays were done in several of the
leukemia cell lines (Fig. 5). PARP protein (Fig. 5A) and
PARP activity levels (Fig. 5B) in MMR-proficient and
MMR-deficient cell lines were quite variable and did not
seem to correlate with MGMT activity. MMR-proficient cell
lines had PARP activity ranging from 45 fmol/μg protein
(AML cells) to 3,430 fmol/μg protein (HEL AML cells).
Although leukemia cells with MMR deficiencies (MSI unstable) tended to have higher PARP activity (510–2,050
fmol/μg protein) than MMR-proficient cells, the difference
between MMR-proficient and MMR-deficient leukemia cell
lines was not statistically significant (P = 0.76, Student's
t test).
Overall, ABT-888 inhibited PARP activity by 78% to
97% (Fig. 5B). The degree of PARP inhibition was roughly
proportional to the degree of ABT-888 potentiation of TMZ
in most leukemia cell lines. For example, ABT-888 induced
90% PARP inhibition in the MMR-deficient cell line HSB2,
which showed the greatest degree of ABT-888–induced
TMZ enhancement (21-fold). Residual PARP activity in
HSB2 was only 60 fmol/μg protein. In contrast, in the
MMR-proficient JM1, which showed less potentiation
(3-fold), ABT-888 provided only 78% inhibition of PARP
activity and the highest residual PARP activity (360 fmol/μg
protein) was observed.
Notably, ABT-888 did not potentiate TMZ growth inhibition in the AML cell line U937, a MMR-proficient cell line
with minimal MGMT activity (Fig. 5D). The U937 cell line
expressed very little PARP by immunoblot (Fig. 5A) and
had minimal PARP activity (Fig. 5B), suggesting that ABT888 may be less effective at potentiating TMZ in cells with
very low PARP activity.
In summary, when MGMT activity, PARP activity, and the
PFs were examined as continuous variables, there were no
significant differences in MGMT activity or PARP activity
between the MMR-deficient and the MMR-proficient cell
lines (P = 0.37 and 0.23, respectively, Wilcoxon rank-sum
test). PFs, however, were significantly higher in MMRdeficient cell lines when compared with those with intact
MMR (P = 0.02, Wilcoxon rank-sum test).
ABT-888 Potentiation of TMZ Is Not Due to NHEJ
PARP also participates in forms of DNA double-strand
break (DSB) repair, including both homologous and nonhomologous recombination and a backup pathway of
NHEJ in cells compromised for classic NHEJ (12, 39, 40).
Figure 2.
TMZ and ABT-888
growth inhibition in leukemia cell
lines. MTT cytotoxicity assays were
done with either TMZ (A) or ABT888 (B) at concentrations ranging
from 1 to 2,000 μmol/L. Percentage cell survival is plotted as a
function of drug concentration.
Leukemia cells include the MMRproficient JM1 ( ), U937 ( □ ),
THP1 (◊), HEL ( ), Raji ( , dotted
line), Daudi ( , dotted line), and
HL-60 ( ) and the MMR-deficient
Jurkat (♦), HSB2 (▴), Reh ( ), and
Molt4 ( ) cell lines. Open symbols,
AML cell lines; closed symbols, ALL
cell lines.
○
▾
• •
▪▵
▪
Mol Cancer Ther 2009;8(8). August 2009
Molecular Cancer Therapeutics
Figure 3. ABT-888 potentiation of TMZ
in MMR-proficient (A) or MMR-deficient
( B ) cell lines. A, AML cell lines THP1
(S1, left), HL-60 (S2, middle), and KG1
(S3, right) treated with TMZ alone ( ) or
TMZ in combination with 0.5 μmol/L ( )
or 5 μmol/L ( ) of ABT-888. Similar results
were obtained with ALL cell lines JM1
(S1; Fig. 2D), Raji, and Daudi (S2; data
not shown). B, T-cell ALL cell lines HSB2
(S1), Molt4 (S2), and pre-B ALL cell line
Reh (S3) treated as above. Cell lines have
low (S1, U1), average (S2, U2), or elevated (S3, U3) MGMT activity. C, summary
of PFs (IC50 TMZ/IC50 TMZ + ABT-888)
in leukemia cell lines that are MMR proficient (MSI stable; S) and MMR deficient
(MSI unstable; U) treated with TMZ combined with either 0.5 μmol/L (□) or
5 μmol/L ( ) of ABT-888. Coefficients of
variation for the modeled IC50s varied from
8% to 29%.D and E, MMR-proficient preB ALL cell line JM1 (D) and MMR-deficient
T-cell ALL cell line Molt4 (E) treated with
TMZ alone ( ) and TMZ in combination
with 0.5 μmol/L ABT-888 (□), 1 μmol/L
O6-BG (◊), or both 0.5 μmol/L ABT-888
and 1 μmol/L O6-BG ( ).
○
▪
•
▪
○
▪
To determine whether PARP potentiation of TMZ was
affected by NHEJ in leukemia cell lines, we examined
the expression of Ku70, which was strongly expressed in
all leukemia cell lines tested, including U937 (Fig. 5A).
To further determine whether PARPi potentiation of TMZ
was affected by NHEJ status, we measured NHEJ activity in
leukemia cell lines using an in vitro NHEJ assay (35). Leukemia cell lysates were incubated with a radiolabeled probe
designed to dimerize as the results of NHEJ as shown in
293T cells (Fig. 5E). MMR-proficient cell lines with similar
ABT-888 potentiation of TMZ had different NHEJ activity
levels. The AML cell lines THP1 and HL-60 had undetectable NHEJ, whereas in the pre-B ALL JM1 cell line NHEJ
was robust. We also examined phosphorylated histone
Mol Cancer Ther 2009;8(8). August 2009
H2AX, which is increased in response to unrepaired DSBs,
and found no correlation between H2AX protein and ABT888 potentiation of TMZ (Fig. 5A). These data suggest that
ABT-888 potentiation of TMZ is not related to its role in
NHEJ.
ABT-888 Potentiation of TMZ in Primary Leukemia
Cells Is Related to Leukemia Subtype
Because leukemia cell lines have developed multiple
adaptations to growth in culture, we also examined the
ability of ABT-888 to potentiate TMZ in primary acute leukemia cells obtained directly from patients (Fig. 6). We determined whether ABT-888 potentiation correlated with
MGMT activity or, in a subset of patients, with MMR status (Table 2). ABT-888 did not significantly potentiate TMZ
2237
2238 ABT-888 and Temozolomide in Acute Leukemia
activity in primary ALL leukemia cells (Fig. 6A; Table 2)
but enhanced the growth-inhibitory effects of TMZ in
two of four AML leukemias (Fig. 6B; Table 2). The lack
of ABT-888 potentiation of TMZ in two primary AML specimens did not seem to correlate with MGMT expression,
as patient specimen p115, which showed the greatest
ABT-888–induced potentiation of TMZ among the primary leukemias (4.6-fold), had elevated MGMT activity
(2,580 fmol/mg protein). All tested primary leukemia cells
were MMR proficient.
To determine whether ABT-888 potentiation of TMZ was
related to baseline PARP activity, we measured PARP activity in 14 primary leukemia samples (Fig. 6C). Similar to leukemia cell lines, primary leukemia cells had widely variable
PARP activity, ranging from <40 fmol/μg protein (below the
level of quantitation) to 1,860 fmol/μg protein. There
seemed to be little correlation between PARP activity and
ABT-888 potentiation of TMZ. Of interest, one of the two
AML primary samples that showed no ABT-888 potentiation of TMZ (sample 4) also showed no PARP activity similar to the U937 cell line.
Discussion
In this study, we used a panel of leukemia cell lines to assess
the relative effects of MGMT activity, MMR status, and
PARP activity on the ability of ABT-888 to potentiate the
antitumor effects of TMZ. Although our data indicate that
ABT-888 was most effective in potentiating TMZ in MMRdeficient cell lines with low MGMT activity, ABT-888 also
potentiated TMZ in MMR-proficient cell lines. In primary
acute leukemia cells, ABT-888 potentiation of TMZ seemed
to correlate only with leukemia subtype, providing >3-fold
potentiation in two of four primary AML samples.
Figure 4.
Effect of MGMT overexpression on ABT-888 potentiation of TMZ in
MMR-proficient and MMR-deficient leukemia cell lines. A, confirmation of increased
MGMT expression by Western blot and
quantitation of increased MGMT expression by densitometry. The MMR-proficient
AML cell line THP1 ( B ) and the MMRdeficient T-cell ALL leukemia cell line
HSB2 (C) were treated with TMZ alone
(○) or TMZ with either 0.5 μmol/L ( ) or
5 μmol/L ( ) of ABT-888. Left, transfected
with pCDNA vector (EV); right, transfected
with pCDNA vector containing the fulllength coding sequence for MGMT.
▪
•
Mol Cancer Ther 2009;8(8). August 2009
Molecular Cancer Therapeutics
Figure 5. Mechanisms of PARP
potentiation in leukemia cell lines.
A, pretreatment PARP, Ku70, and
phospho-H2AX protein expression
in four MMR-deficient (U) and three
MMR-proficient (S) leukemia cell
lines. Expression is normalized to
actin expression. B, PARP activity
for the leukemia cell lines described
in A untreated (white columns) or
treated with either 0.5 μmol/L (gray
columns) or 5 μmol/L (black columns) of ABT-888. PARP activity
(fmol/μg protein) was determined
as described in Materials and Methods. C, PARP activity in established
leukemia cell lines. Horizontal line,
limit of detection for assay. D,
dose-response curve of TMZ alone
( ) and TMZ in combination with either 0.5 μmol/L ( ) or 5 μmol/L ( ) of
ABT-888 or 1 μmol/L O6-BG (×) in
U937 AML cells. E, small-scale
NHEJ in vitro assay. A 3-kb probe,
designed to favor dimerization over
recircularization or multimerization,
was incubated with different leukemia cell line cell extracts. The
presence of robust NHEJ can be detected by the formation of the 6-kb
dimer as shown in the 293T control
lane. Lane 1, DNA molecular weight
ladder, lane 2, radiolabeled DNA
fragment only (no cell lysate); lane
3, NHEJ-proficient control cell line
293T; lanes 4 to 6, leukemia cell
lines THP1, JM1, and HL-60.
○
•
▪
Although Tentori et al. (41) had also noted that leukemia
cell lines could be sensitized to TMZ using the PARPi benzamine, this is the first report to examine the effect of
MGMT activity on the ability of PARPis to potentiate
TMZ in leukemia MMR-proficient and MMR-deficient cell
lines. This is also the first reported examination of ABT888 potentiation of TMZ in primary acute leukemia. Similar
to previous reports in leukemia cell lines, colon cancer, and
glioma, PARPi potentiation of TMZ was more pronounced
in cells with MMR deficiencies (11, 14, 19).
Several PARPis are under investigation in oncology clinical trials. ABT-888 is undergoing phase 1 testing in adults
with melanoma. Other PARPis under clinical study include
AZD2281 (AstraZeneca), AG014699 (Pfizer) in combination
with TMZ for adults with solid tumors and metastatic malignant melanoma (42), INO-1001 (Inotek/Genentech) in
combination with TMZ for metastatic melanoma and glioblastoma multiforme (43), KU59436 (KuDOS/AstraZeneca)
Mol Cancer Ther 2009;8(8). August 2009
in advanced solid tumors (including patients with BRCA1/
BRCA2 mutations; ref. 44), and BS201 (BiPar) for refractory
solid tumors and lymphomas (12). A better understanding
of the factors that govern PARPi potentiation of TMZ may
allow for the prospective determination of which tumors
most likely to respond to this combination.
Prior clinical studies in both adults and pediatrics have
suggested that TMZ may be effective in AML patients with
low/absent MGMT tumor activity but is less effective in the
presence of MMR deficiency or elevated MGMT activity
(3, 4). Our data suggest that PARPi may be effective at potentiating TMZ in AML patients without MMR deficiencies
and that this potentiation may be independent of MGMT
activity. Although common in adults with treatment-related
AML (45), MMR deficiencies are uncommon in pediatric
patients with newly diagnosed AML. However, pediatric
patients with relapsed leukemia frequently have elevated
MGMT (4) and this group of patients would benefit from
2239
2240 ABT-888 and Temozolomide in Acute Leukemia
potentiating agents that could overcome both MMR deficiencies and elevated MGMT activity.
In MMR-deficient cell lines, we found an inverse correlation between MGMT activity and potentiation of TMZ by
ABT-888. MGMT activity seemed less important in determining ABT-888 potentiation of TMZ than MMR status in these
leukemia cell lines. First, we observed 3-fold ABT-888 potentiation of TMZ in MMR-proficient leukemia cell lines and
10-fold ABT-888 potentiation of TMZ in MMR-deficient leukemia cells with elevated MGMT activity (Fig. 3C). Second,
overexpression of MGMT in both MMR-deficient and
MMR-proficient cell lines was unable to completely block
ABT-888 potentiation of TMZ (Fig. 4). PARPi potentiation of
TMZ in the presence of MGMT was also observed using the
PARPi 3AB in colon cancer cell lines (46). These data suggest
that neither increased MGMT activity nor MMR proficiency
precludes the ability of PARPi to potentiate TMZ.
In some cases, the effects of ABT-888 seemed to depend
on a threshold level of PARP activity because ABT-888 did
not potentiate TMZ in either the U937 AML cell line or primary leukemia cells with very low PARP activity (Figs. 5C
and 6A). However, ABT-888 did not result in TMZ potentiation in many primary ALL samples with high PARP activity, indicating that the presence of robust PARP activity is
not sufficient for ABT-888 potentiation. There was also
one AML cell line (KG1) with very low PARP activity, which
showed 3-fold ABT-888 potentiation of TMZ, suggesting
that either ABT-888 potentiation of TMZ may be dependent
on nonribosylation functions of PARP. TMZ potentiation effects that are independent of PARP inhibition (i.e., off-target
effects) cannot be excluded. Previous data showed that the
PARPi GPI-15427 increased the antitumor activity of TMZ
and irinotecan in colon cancer xenografts irrespective of
PARP activity (16). Although the range of PARP activity
Figure 6. PARP activity and ABT-888
potentiation of TMZ in primary leukemia
samples. A to D, dose-response curve of
TMZ alone (○) or TMZ in combination with
either 0.5 μmol/L ( ) or 5 μmol/L ( ) of ABT888 in four representative primary leukemia patient samples. A, representative
ALL patient samples in which ABT-888
did not potentiate TMZ. Left, 11-y-old
Caucasian female with newly diagnosed
pre-B ALL; right, 2-y-old Hispanic female
with newly diagnosed T-cell ALL. Potentiation factors are listed in Table 2. B, AML
patient samples in which ABT-888 potentiated TMZ. Left, 2-d-old Hispanic male with
fulminant AML/myelodysplastic syndrome; right, 17-y-old Hispanic male with
relapsed M0 AML (trisomy 8 translocation).
C, PARP activity in 14 patient leukemia
samples. Horizontal line, limit of detection
for assay.
•
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Mol Cancer Ther 2009;8(8). August 2009
Molecular Cancer Therapeutics
Table 2. Summary of MGMT activity, PARP activity, and ABT-888 potentiation of TMZ in primary pediatric cells
Cell ID
Leukemia subtype
MSI
MGMT activity (fmol/mg protein)
PARP activity (fmol/μg protein)*
ABT-888 PF†
p115
p151
p120
BM1261
p145
p152
p157
p158
BM1169
BM1339
BM1350
p153
p154
AML
AML
AML
AML
Pre-B ALL
Pre-B ALL
Pre-B ALL
Pre-B ALL
Pre-B ALL
Pre-B ALL
Pre-B ALL
T-ALL
T-ALL
S
ND
S
ND
ND
S
S
ND
ND
ND
ND
ND
ND
2,580
1,200
UD
151
1,690
325
1,260
3,250
310
1,110
550
1,580
1,760
ND
670
370
UD
1,320
350
1,180
1,860
40
90
70
430
310
4.6
3.2
1.2
1
1
1
1
1
1
1.2
1.5
1
1
Abbreviations: S, MSI stable; ND, not determined; UD, undetectable.
*MGMT activity <100 fmol/mg protein; PARP <40 fmol/μg protein.
†
IC50 TMZ/IC50 TMZ + ABT-888. Value of 1 indicates no potentiation.
in most primary tumors is unknown, Plummer et al. (47)
examined PARP activity in adult melanoma specimens
and noted that PARP activity was quite variable, ranging
from 1.7 to 3,600 pmol PARP/mg protein. Hence, it is likely
that the effectiveness of PARPi will vary, reflecting variability in both baseline PARP activity and the efficiency of
PARP inhibition.
Although best known for its role in BER, PARP also participates in DSB repair (5). Evidence suggests that PARP
competes with Ku for DSBs and PARP can either preserve
DSBs for homologous recombination (48) or (in the absence of Ku) initiate the use of a backup NHEJ repair pathway involving DNA ligase III (40). The effects of PARP
inhibition could be influenced by the intactness of the
DSB repair pathways; for instance, in the absence of Ku
or BRCA1/2, tumor cells may be very sensitive to PARP
inhibition (49). We have shown here that NHEJ status
did not affect the ability of ABT-888 to potentiate TMZ
in leukemia cell lines. Further study is needed to determine whether the status of the backup NHEJ repair pathway or homologous recombination influences ABT-888
potentiation of TMZ.
The influence of MGMT activity, PARP activity, and MSI
on the ability of ABT-888 to potentiate TMZ is derived from
a limited number of leukemia cell lines and therefore should
be interpreted with caution. PARPi potentiation of TMZ
in MMR-proficient cell lines may also be limited clinically
because ABT-888 potentiation is seen only at high TMZ
concentrations (>100 μmol/L). PARPi potentiation of TMZ
could also be influenced by factors that were not examined,
such as ABT-888 transport or metabolism.
In limited phase 1 studies of TMZ in patients with leukemia, where patients may have either ALL or AML, clinical responses have been observed exclusively in patients
with AML. In a phase 1 study of TMZ in pediatric leukemia (4), two of eight patients with AML had objective
responses. These patients had absent or low MGMT activity
and no MSI. Similarly, in an adult phase 1 study of TMZ
Mol Cancer Ther 2009;8(8). August 2009
in acute leukemia, the four patients with an objective
response to TMZ (complete remission or complete remission with incomplete platelet recovery) had AML (3). In
this study, in vitro ABT-888 potentiation of TMZ was
seen in some primary samples from patients with AML
(Table 2). Although the reason for this is unclear, it may
be that more complete PARP inhibition can be obtained
in patients with AML.
In summary, we have shown that the PARPi ABT-888 can
effectively enhance the activity of TMZ, potentiating TMZ
growth inhibition 3- to 21-fold in leukemia cell lines. ABT888 potentiation of TMZ was most effective in cells with
MMR deficiencies and low MGMT activity. Unexpectedly,
PARP inhibition also potentiated TMZ activity in MMRproficient leukemia cell lines, providing a 3- to 7-fold
enhancement of TMZ growth inhibition independent of
MGMT activity. Our data suggest that ABT-888 may
function as a useful potentiator of TMZ; further studies,
particularly in AML, are warranted.
Disclosure of Potential Conflicts of Interest
M.E. Dolan: coinventor of O6-BG, which has been licensed to Access
Oncology, Inc. No other potential conflicts of interest were disclosed.
Acknowledgments
We thank Gouqing (Mary) Ge for excellent technical assistance and MengFen Wu for statistical support.
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